Isomeric Separation of Permethylated Glycans by Porous Graphitic Carbon (PGC)-LC-MS/MS at High Temperatures

Abstract

Permethylation is a common derivatization method for MS-based glycomic analyses. Permethylation enhances glycan ionization efficiency in positive MS analysis and improves glycan structural stability. Recent biological glycomic studies have added to the growing body of knowledge and suggest the need for complete structural analysis of glycans. However, reverse phase LC analysis of permethylated glycans usually results in poor isomeric separation. To achieve isomeric separation of permethylated glycans, a porous graphitic carbon (PGC) column was used. PGC columns are well-known for their isomeric separation capability for hydrophilic analyses. In this study, we have optimized temperature conditions to overcome the issues encountered while separating permethylated glycans on a PGC column and found that the highest temperature examined, 75 °C, was optimal. Additionally, we utilized tandem MS to elucidate detailed structural information for the isomers separated. Glycan standards were also utilized to facilitate structural identifications through MS/MS spectra and retention time comparison. The result is an efficient and sensitive method capable of the isomeric separation of permethylated glycans. This method was successfully applied for the isomeric characterization of N-glycans released from the breast cancer cell lines MDA-MB-231 and MDA-MB-231BR (brain seeking). A total of 127 unique glycan structures were identified with 39 isobaric structures, represented as 106 isomers, with 21 nonisomeric glycans. Thirty seven structures exhibited significant differences in isomeric distribution (P < 0.05). Additionally, alterations in the distribution of isomeric sialylated glycans, structures known to be involved in cell attachment to the blood-brain barrier during brain metastasis, were observed.

Figure 1

Extracted ion chromatograms (EICs) of reduced and permethylated glycan structures of (A) Man 7, (B) Man 8 (C) Hex₅HexNAc₄NeuAc₂ and (D) Hex₆HexNAc₅NeuAc₂ derived from Ribo B (A and B) and bovine fetuin (C and D), which were separated using a PGC column at ambient temperature (25°C), 40°C, 55°C, and 75°C. Symbols: , N-acetylglucosamine (GlcNAc); , Galactose (Gal); , Fucose (Fuc); , Mannose (Man); , N-acetylneuraminic acid (NeuAc/Sialic Acid)

Figure 2

(A) EIC of glycan F1A2G1 from mixed standard glycan samples, which were chemically synthesized structures. (B) CID MS/MS spectrum of branch fucosylated glycan that was eluted at 29.2 min. (C) CID MS/MS spectrum of core fucosylated glycan that eluted at 32.7 min. Symbols: as in Figure 1.

Figure 3

(A) EIC of glycan F1A2G1 with galactose attached to different branches, which were released from human blood serum (B) CID MS/MS spectrum of isomeric structure eluted at 32.8 min, with molecular modeling of the structure. (C) CID MS/MS spectrum of isomeric structure eluted at 34.1 min, with molecular modeling of the structure. Symbols: as in Figure 1.

Figure 4

EICs of (A) bi-sialylated glycans and (B) Hex₃HexNAc₃ glycans released from the 231 cell line and the 231BR cell line. Distributions of isomeric glycans in the 231 and 231BR cell lines exhibited significant differences (P<0.05). Symbols: as in Figure 1.